This application is a National Stage entry of International Application No. PCT/EP00/04372, filed May 16, 2000, the entire specification claims and drawings of which are incorporated herewith by reference.
The present invention generally relates to the field of organic chemistry.
More particularly, the invention relates to a process for the preparation of Cefuroxime acid, i.e. (6R,7R)-7-[[2-furanyl(sin-methoxyimino)acetyl]amino]-3-carbamoyloxymethylceph-3-em-4-carboxylic) acid, and the salts thereof, starting from (6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-hydroxymethyl-ceph-3-em-4-carboxylic acid (deacetyl 7-glutaryl ACA).
Cefuroxime acid is a key intermediate for the industrial synthesis of two third generation cephalosporins, Cefuroxime sodium (for the injection administration) and Cefuroxime Axetil (for the oral administration). These molecules are therapeutically valuable thanks to their effective broad spectrum antibacterial activity against gram-negative bacterials, in particular in the treatment of immunodepressed patients. Their effectiveness is advantageously combined with remarkable resistance to xcex2lactamases.
The synthesis of Cefuroxime disclosed in U.S. Pat. No. 3,966,717 and U.S. Pat. No. 3,974,153 comprises 8 synthetic steps starting from 7-ACA (7-amino cephalosporanic acid). Such high number of steps, which causes a low overall yield, is due to the introduction of two protective groups, the first (e.g. thienyl acetyl) on the amine function and the second (e.g. benzhydryl) on the 7-ACA acid function.
Subsequently, processes starting from 7-ACA have been developed (Wilson, E.M. Chemistry and Industry 1984, 217) which do not involve the use of protective groups and remarkably reduce the number of steps. In particular, the best process, illustrated in Scheme 1, comprises 3 steps:
1. Conversion of 7-ACA into deacetyl-7-ACA;
2. Acylation of the amino group;
3. Carbamoylation of the C-3 alcohol group (Scheme 1). 
It has now surprisingly been found that Cefuroxime can be prepared starting from intermediates of 7-ACA enzymatic synthesis without isolating any intermediate.
The enzymatic synthesis of 7-ACA involves, depending on the used process, an intermediate that can either be glutaryl-7-ACA (U.S. Pat. No. 5,424,196; Bianchi, D., Bortolo, R., Golini, P., Cesti, P. La Chimica e l""Industria 1998, 80, 879), which can be enzymatically converted into deacetyl 7-glutaryl ACA (II), or (II) itself, from fermentative processes yielding des-Cephalosporin C (U.S. Pat. No. 4,533,632).
The process object of this invention, illustrated in Scheme 2, comprises the following steps:
1. Extraction of deacetyl 7-glutaryl ACA (II) aqueous solution at acid pH with organic solvents (for example according to the procedures disclosed in U.S. Pat. No. 5,801,241).
2. Drying the resulting solution while preventing lactonization of the intermediate.
3. Carbamoylation of the hydroxymethyl group at the 3-position by reaction with chlorosulfonyl isocyanate or similar products.
4. Extraction of the carbamoyl derivative from step 3 with water at neutral pH.
5. Enzymatic hydrolysis of the amide at the 7-position of the cephalosporanic ring with glutaryl acylase.
6. Acylation of the amino group by condensation with 2-furanyl(sin-methoxyimino)acetic acid chloride or mixed anhydride. 
The process of the invention involves a reduction of the number of the steps compared with the known processes for the preparation of Cefuroxime, as it requires neither the protection of the carboxyl at the 4-position of the cephalosporanic ring nor that of the amino group at the 7-position nor the recovery of any intermediates, thus causing a remarkable increase in the overall yield directly starting from des-Cephalosporin C or Cephalosporin C fermentation broth after enzymatic deacetylation.
Furthermore, the process of the invention allows making use of intermediates, which, contrary to 7-ACA, are particularly stable in aqueous solution.
The process according to the present invention provides Cefuroxime acid or a salt thereof which can be transformed into the corresponding commercial products Cefuroxime sodium and Cefuroxime axetil.
The process described above comprises the preparation of an intermediate, which has to day never been described, namely (6R,7R)-7-[(4-carboxy-1-oxobutyl)amino]-3-carbamoyloxy-methyl-ceph-3-em-4-carboxylic acid of formula (III) 
or a salt thereof.
The steps of the process according to the present invention for the preparation of Cefuroxime acid starting from deacetyl 7-glutaryl ACA (II) are described in detail hereinbelow.
1. Extraction of a deacetyl 7-glutaryl ACA (II) aqueous solution with an organic solvent, preferably cyclohexanone (U.S. Pat. No. 5,801,241).
A deacetyl 7-glutaryl ACA aqueous solution, at a concentration ranging from 1 to 20%, is adjusted to pH ranging from 1.0 to 3.0, preferably 1.5, at temperatures ranging from 0 to 15xc2x0 C., preferably from 0 to 5xc2x0 C. These conditions prevent degradation of the substrate to give a lactone following condensation of the carboxyl at the 4-position with the hydroxyl bound to the methyl at the 3-position. The resulting solution is added with 0.5 to 2 volumes of an organic solvent, preferably cyclohexanone, and extraction is carried out at temperatures ranging from 0 to 5xc2x0 C. Phase are separated, then the aqueous phase is back-extracted with 0.5÷1.0 volumes of solvent and the organic phases are combined.
2. The resulting organic solution is brought to temperatures from 0 to 15xc2x0 C., preferably from 0 to 5xc2x0 C., adjusted to apparent pH ranging from 6 to 8, preferably 7, with a solution of triethylamine in the organic solvent used for the extraction. The resulting mixture is concentrated in vacuo and at a temperature below 25xc2x0 C., to obtain a suspension with a water content below 0.5%, which is then processed in the subsequent step.
3. Conversion of deacetyl 7-glutaryl ACA (II) into the corresponding 3-carbamoyloxymethyl derivative (III), by reaction in cyclohexanone with an activated isocyanate, preferably chlorosulfonyl isocyanate. The suspension isolated at the end of the previous step, having a concentration ranging from 1 to 10%, is cooled to temperatures ranging from xe2x88x9230 to 0xc2x0 C., preferably xe2x88x9210xc2x0 C., and added, in small portions, with 1÷5 mols of chlorosulfonyl isocyanate per mol of substrate. The resulting heterogeneous mixture is then kept at such temperature until completion of the reaction, whose progression is monitored by HPLC chromatography.
4. Extraction of the carbamoyl derivative obtained in step 3. The solution from step 3 is added with 0.1÷0.3 volumes of cold water and the resulting heterogeneous mixture is then adjusted to pH ranging from 6 to 8, preferably 7, at temperatures ranging from 0 to 15xc2x0 C., preferably from 0 to 5xc2x0 C., with an aqueous ammonia solution. The two phases are separated; the organic phase is back-extracted with water, to 0.2 volumes; the aqueous phases are combined and the resulting solution, having a concentration ranging from 5 to 30%, is processed in the subsequent step.
5. Conversion of the 7-glutaryl 3-carbamoyloxymethyl derivative (III) into the corresponding 7-xcex2-amino derivative (IV) by enzymatic hydrolysis of the amide at the 7-position of the cephalosporanic ring with glutaryl acylase. The resulting solution from the previous step is added with a glutaryl acylase isolated from an Escherichia Coli culture, suitably supported on a macroreticular resin, preferably polyacrylic epoxide, and the resulting suspension is kept at pH 7.0-9.0, preferably 7.5, at temperatures ranging from 20 to 30xc2x0 C., preferably 25xc2x0 C., until completion of the reaction. The progression of the hydrolysis of the glutaryl derivative to the corresponding cephalosporanic ring is monitored by HPLC chromatography. The reaction yield is higher than 85%. After completion of the reaction, the enzyme is removed and the product is converted into Cefuroxime acid.
6. (6R,7R)-7-Amino-3-carbamoyloxymethylceph-3-em-4-carboxylic acid (IV) is directly converted into Cefuroxime acid (I), adding the aqueous solution with a concentrated methylene chloride solution of 2-furanyl(sin-methoxyimino)-acetic acid chloride or mixed anhydride (preferably the chloride).
Optionally, intermediate (IV) can be recovered by acidification to isoelectric pH of the solution from the previous step, then be worked in the acylation of the amino group at the 7-position of the cephalosporanic ring, by following the method described in example 2 of U.S. Pat. No. 3,974,153.